The Effect Of Quinoxaline1,4-dioxides (QDNO’S) On The Expression Of Genes And Proteins In Sensitive Escherichia Coli

Quinoxaline compounds is a class of synthetic antibacterial agents, which are derivatives of quinoxaline-1,4-dioxides and showing great antimicrobial and growth-promoting effect. In vitro antibacterial test of QdNOs and preventive effect research of QdNOs on livestock diseases caused by common pathogenic bacteria have been carried out in our lab. The results indicate that QdNOs do have high antibacterial effect, but antibacterial mechanism of QdNOs is still unclear except for the research on quindoxin, which has been proved to inhibit bacterial DNA synthesis. The genuine mechanism of DNA damage and antibacterial targets need to be more clearly researched and discussed. Today, genomic and proteomic technologies have become the effective methods for the study of mechanism of drug actions and targets. Cyadox, as a new member of QdNOs, and enteric pathogenic Escherichia coli that is sensitive to cyadox were selected as objects of this study, and olaquindox was selected as the old member of QdNOs drug. Differentially expressed genes in E. coli treated with cyadox or olaquindox were identified by gene chip, then those differential genes directly connecting with the drug and the effect of the drugs on bacteria’s growth, reproduction, metabolism were analyzed. Hypothesis of antibacterial mechanism and the possible target protein of the drug in bacteria were drawn based on the previous analysis. To verify and supplement the differentially expressed genes, differentially expressed proteins were identified by the optimized E. coli two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). The study of antibacterial mechanism of cyadox would speed up the development pace of the the quinoxaline compounds, provide more comprehensive information for new drug application in line with international and national standards, provide a reference for future clinical use, and guarantee the safety for the humen and animals.1. Bacterial selection and drug treatment concentration and timeIn order to select the appropriate sensitive strains, minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of cyadox and olaquindox in pathogenic E. coli were performed by a broth microdilution method, under aerobic and anaerobic conditions respectively. The MICs of Escherichia coli C84010against cyadox and olaquindox were determined to be1μg/mL and4μg/mL in anaerobic conditions, and the MIC of E. coli C84010against two drugs were all determined to be16μg/mL. Since it was the most sensitive to cyadox under anaerobic environment compared to other strains, E. coli C84010was selected as subject for our research. The result showed that the strain’s susceptibility for cyadox and olaquindox was much higher in anaerobic environment in consistence with the existing literatures on the anaerobe-selective activity of quinoxalines, indicating that it was more meaningful to explore the antibacterial mechanism of cyadox under anaerobic condition.To find out the genes and proteins directly connected with the drug and the relationship between concentration of the drug and the change of differentially expressed genes, the growth curve of E. coli treated with different concentrations of drug was determined by viable count method. The results showed that the growth of E. coli which was treated with cyadox (0.5μg/mL) or olaquindox (4μg/mL) was decreased significantly, but the curve was still on the rise; and after being treated with cyadox (4μg/mL) and olaquindox (16μg/mL) for30min,90%of the bacteria were killed. So the drug concentrations were fixed as0.5and4μg/mL for cyadox,4and16μg/mL for olaquindox, and drug incubation time was fixed as30min in later experiments.2. The differentially expressed genes for E. coli with treatment of cyadox and olaquindoxTo better understand the molecular antibacterial mechanism, E. coli treated with cyadox (0.5and4μg/mL) and olaquindox (4and16μg/mL) for30min were used as four drug treatment groups, and E. coli incubated with1%DMSO for30min was used as the control group in accordance with the above results. The gene chip was adopted to identify differentially expressed genes in drug treated E. coli, afterwards gene function classification and protein network analysis were carried on.25differentially expressed genes were screened out from the E. coli treated with cyadox (0.5μg/mL), which were all significantly up-regulated;55differentially expressed genes were selected from E. coli treated with cyadox (4μg/mL),46of which were significantly up-regulated and9were significantly down-regulated. In these differentially expressed genes from the E. coli treated with laquindox (4μg/mL),68and98genes showed significant upregulation and downregulation, respectively;102differentially expressed genes were filtered out from E. coli treated with olaquindox (16μg/mL), of which81were significantly up-regulated and21significantly reduced.These differentially expressed genes could be divided into seven categories. They are the SOS genes, cell membrane-related genes, cytotoxic genes, substance synthesis am metabolism-related genes, cytochrome genes, DNA transcription regulatory and putative genes. Those up-regulated SOS genes were common in the four drug treatment groups. The SOS genes were accounted for80%of the total differentially expressed genes from E. coli treated with cyadox (0.5μg/mL), and the change folds of these genes were increased significantly when the dose of the drugs rose. It was shown that cyadox and olaquindox all had an obvious DNA damage effect. It was inferred that the radical intermediate of cyadox reduction under anaerobic condition was responsible for the poison effect of DNA C1, which bringed about DNA double stand breaks and other DNA damages in E. coli.Higher concentration of Cyadox or olaqiundox caused90%cell death from the serious DNA damage, and the survival bacteria switched on some stress-resistant regulatory genes (yjbJ, mar A), which regulated the expression of multiple genes adapted to rapid environmental change, such as slowing down protein synthesis speed (yafO), shutting down the switch of the flagella (fliM), and weakening membrane transport of substances (rbsC, proV, and treB), also upregulating efflux pump gene (ydhC) that excluding cyadox outside. 3. The differentially expressed proteins for E. coli with treatment of cyadox and olaquindoxIn order to complement and verify the results of the differentially expressed genes in translation level, optimized2D-PAGE system of E. coli was used and differentially expressed proteins were screened from bacteria treated with cyadox and olaquindox. The main functions of the differentially expressed proteins were DNA repairing, anti-oxidative stress, protein synthesizing, controlling of bacterial morphology and bacterial toxicity. LexA protein was down-regulated but in gene chip the lexA gene was up-regulated in contrast. This result was based on self-cleavage activity of LexA, after which the expression of SOS genes were activated. At the protein level, some other oxidative stress proteins like SSB and YgfZ were found up-regulated to protect bacterial DNA against oxidative damage. Proteins functioning as synthesis elongation factor were down-regulated in consistent with the upregulation of protein translation suppressor genes in gene chip, speculating that bacteria turned off protein synthesis until the resumption of normal growth. Besides, the MreB protein which determined E. coli shape was down-regulated, suggesting the change of the bacterial morphology. This was in accordance with up-regulated expression of sulA in microarray, which leaded to the filamentation of bacteria.In conclusion, this study found out a large number of DNA repair genes and proteins in mRNA and protein levels, indicating that the reductive activation of cyadox and olaquindox under anaerobic conditions cause serious damage to the bacterial DNA and interfere with DNA replication, thus playing the antibacterial action. Bacterial DNA damage that was induced by the quinoxalines also directly or indirectly affect all aspects of bacterial metabolic activity. Therefore, the research provided a comprehensive and systematic theoretical basis for further study of QdNOs antibacterial targets and antibacterial pathways.